Protocol

Fluorescent In Situ Hybridization (FISH) on Diploid Nuclei and Mitotic Chromosomes from Drosophila melanogaster Larval Tissues

¹ Université de Toulouse, Université Paul Sabatier, Laboratoire de Biologie Moléculaire Eucaryote, F-31000 Toulouse, France
² Centre National de la Recherche Scientifique, Laboratoire de Biologie Moléculaire Eucaryote, F-31000 Toulouse, France

³ Present address: Adolf Butenandt Institut, Molekularbiologie, 80336 Münich, Germany.

⁴Corresponding author (Roxane.Blattes{at}med.uni-muenchen.de).

INTRODUCTION

This protocol adapts the multicolor fluorescent in situ hybridization (FISH) method for use with interphase nuclei and mitotic chromosomes of neuroblasts from dissected Drosophila melanogaster larval brains. It is suitable for the simultaneous detection of two or more target sequences, is readily applicable to heterochromatin sequences, and provides the specificity and sensitivity needed to covisualize single-copy and repeated sequences. It is equally successful for detecting other sequences, such as single-copy euchromatin genes. This is made possible chiefly through the use of fixation procedures adjusted to suit the nature of the hybridization targets (e.g., condensed heterochromatin sequences), as well as the use of a mild chemical treatment to denature DNA instead of heat. Also, omitting mitotic blockers such as colchicine yields chromosomes with well-preserved morphology and permits detailed mapping of detected sequences. Most importantly, using this technique on diploid mitotic chromosomes and interphase nuclei allows the unambiguous localization of under-replicated heterochromatin sequences that cannot be resolved within the chromocenter of polytene chromosomes.